One-Step Rapid and Sensitive ASFV p30 Antibody Detection via Nanoplasmonic Biosensors

ABSTRACT African swine fever (ASF) is one of the most serious transnational swine diseases in the world. The case fatality rate of susceptible pigs is up to 100%. Currently, no commercial vaccine is available, so the prevention and control of ASF mainly relies on early diagnosis and culling of infected pigs. As the ASF virus continues to evolve, develop, and diversify, nucleic acid testing becomes less efficient. Here, we developed a method for the rapid and direct optical measurement of African swine fever virus (ASFV) antibody in vitro. This one-step procedure requires nearly no sample preparation and involves p30 protein-specific label-free integration into standard 96-well plates. Using a nanoplasmonic biosensor with extraordinary optical transmission (EOT) effect, one-step sample addition, ASFV antibody was detected within 20 min. The positive antibody showed a satisfactory sensitivity and linear relationship in the dilution ratio of 1:100–1:16000. It was used for the detection of clinical serum samples with a coincidence rate of 96.6%. The measurement results can be automatically analyzed and displayed on a conventional microplate meter computer and connected device. Our detection method can be widely applied in point-of-care testing (POCT) of ASFV antibody in pig farms. IMPORTANCE African swine fever (ASF) is a serious transnational disease caused by the African swine fever virus (ASFV), which is highly contagious in wild boars and domestic pigs. There is currently no available vaccine for ASF; therefore, development efforts are a key priority as ASFV continues to evolve and diversify. The ASF antibody rapid detection platform comprising the nanoplasmonic biosensor with extraordinary optical transmission effect can greatly reduce the detection time and improve detection flux while maintaining detection sensitivity and specificity. The one-step sample addition can effectively avoid cross contamination of samples in the detection process. The detection method provides a solution for the rapid and accurate real-time monitoring of ASF in pig farms.


Reviewer #2 (Comments for the Author):
In the article titled "One-step rapid and sensitive ASFV P30 antibody detection via Nanoplasmonic sensors" the authors have described the development and validation of new point of care tool for the diagnosis of ASF. However, few points needs to be addressed related to the development and validation of the assay for the improvisation of the manuscript. 1.Line no. 60, The authors mention the low efficiency in detection of nucleic acid. Provide the justification and citation for the same.
2.The introduction section should described about the detection system of antibodies through nanoplasmonic biosensors. 3. Line no. 98. It is not clear whether these 164 pig samples were positive or suspected ASF specimens? 4. Line no. 99-100. The SPF free serum samples must have been used as negative control. Mention the same for better understanding of the reader. 5. Whether the animal ethical approvals for the study in experimental materials was taken? 6. Several other proteins such as p54, p72, p22 have shown to induce antibody response in pigs. Why specifically p30 was chosen to develop the assay? 7.Line no. 116-117. The reference standard test utilized in this study was ASFV p72 ELISA. However, the assay developed is based on p30 which is not appropriate to make comparison of sensitivity of the assay. 8. Line no. 141. How the mold and chip has been formed and made into 96 well plate format is not clearly explained. 9.Line no. 164. What is the detection reagent used for the assay? 10.Line no.162-175. The principle of binding of the immunological moieties is not clearly explained, even the detection system is not effectively written i.e, chromogenic, luminex. 11.Line 224-255. Authors have not explained the limit of detection of the assay, sensitivity and specificity with reference to gold standard.

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September 20, 2022
Dear Editors and Reviewers, Thank you for your letter and for giving us the opportunity to revise our manuscript entitled "One-step rapid and sensitive ASFV p30 antibody detection via Nanoplasmonic biosensors" (Spectrum02343-22).Those comments and suggestions are all valuable and helpful in revising and improving our manuscript, as well as providing an important guidance to our research. We have studied the comments carefully and have made corrections which we hope will meet with approval. The revisions are marked in blue in the revised manuscript. Additionally, these changes will not influence the content and framework of the paper. We have modified the manuscript accordingly, and the detailed corrections are listed point by point as follows: The comments of reviewer 1 and corresponding responses.
Comment # 1: The cut-off value should be determined using more negative sera against ASFV. Response: We are so grateful for your suggestion. Accordingly, in order to further determine the cut-off values, we added 20 negative serum samples on Line no. 105, the cut-off value and ROC curve in figure4a and Figure4b has also been modified accordingly.
Comment # 2: The developed method should be evaluated using more sera (strongly/weakly positive or negative) samples with a clear background. Response: Thank you for your comment. As you say, in order to further evaluate the developed method, on the basis of 166 clinical sera samples, we added 20 clinical sera samples. The strength (strongly/weakly positive or negative) of the 186 clinical sera was assessed using the OIE-recommended ASFV VP72 Antibody ELISA kit (INGEZIM 11.PPA.K3; Spain), the blocking rate represents the strength of serum (blocking rate%, X%≥50%,ASFV positive; X%≤40%,ASFV negative; 50%≥X%≥ 40%,Suspected samples). Among them, there are 47 sera samples with the blocking rate higher than 90%, 68 sera samples with the blocking rate between 60%-80%, 31 sera samples with the blocking rate between 50%-60%, and 40 sera samples with the blocking rate lower than 40% .Details are provided in Supplemental Table S1. Comment # 3: The claimed "one-step, rapid, and sensitive" detection should be justified and discussed. Response: Thank you for this valuable suggestion. We have suitably incorporated this in the discussion section according to your request on Line no. 295-305. Regarding the "one-step, rapid, and sensitive" advantages of the Nanoplasmonic biosensor compared to the traditional ELISA method, the Nanoplasmonic biosensor method showed comparable sensitivity with indirect immunofluorescence in the dilution range of 1:100-1:16000 Figure S1, it was used for the detection of clinical serum samples with a coincidence rate of 96.6 %. In addition, as shown in Figure 1b, the method has the advantages of one-step sample addition, automatic analysis, and fast generation of results. For further improvement, a computer and application-controlled microplate reader can be developed, so it can output stable detection results within the total reaction time of 20 min; the detection time can thereby be shortened to the greatest extent.
Comment # 4: The manuscript was poorly written and should be revised by native English speakers. Response: Thank you for pointing this out. We apologize for the poor language of our manuscript. We worked on the manuscript over a long period of time, and the repeated addition and deletion of sentences and sections have evidently led to poor readability. We have now worked on both language and readability and also recruited native English speakers for language corrections. We sincerely hope that the flows of ideas and language clarity have been substantially improved.

The comments of reviewer 2 and corresponding responses.
Comment # 1: Line no. 60, The authors mention the low efficiency in detection of nucleic acid. Provide the justification and citation for the same. Response: Thank you for raising this question. According to your suggestion, we have cited reference (8) in support of this statement.
Comment # 2: The introduction section should described about the detection system of antibodies through nanoplasmonic biosensors. Response: We highly appreciate the reviewer for this insightful suggestion. The following text has been added in the Line no. 85-90: Specifically, Goat anti-pig or other host IgG was fixed on the chip surface, and the remaining sites were blocked with BSA. After the initial OD value was detected, serum samples and antigen labeled with gold particles were added in the wells in a one-step way, forming the Goat anti-host IgG-detect antibody-antigen sandwich complexes, the microplate reader captures the refractive change due to the complex at the bottom of the Nanoplasmonic biosensors and displays it on the compute. Comment # 3: Line no. 98. It is not clear whether these 164 pig samples were positive or suspected ASF specimens? Response: Thank you for your question. We are deeply sorry for the misunderstanding caused by the previous incorrect description. I have revised line no. 103-105.These sera are all clinically suspected samples. Comment # 4: Line no. 99-100. The SPF free serum samples must have been used as negative control. Mention the same for better understanding of the reader. Response: We are so grateful for this question. As you say, In fact, we are using SPF serum as the negative control, but due to the misunderstanding caused by the wrong description, we has been revised in Lines 103-106. Comment # 5: Whether the animal ethical approvals for the study in experimental materials was taken? Response: We are so grateful to you for raising this important question. All animal experiments materials were reviewed, approved, and supervised by the Institutional Animal Care and Use Committee at the Huazhong Agricultural University (ID Number: 202208220001), and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Research Ethics Committee, Huazhong Agricultural University, Hubei, China.We have included this information in the revised manuscript.
Comment # 6: Several other proteins such as p54, p72, p22 have shown to induce antibody response in pigs. Why specifically p30 was chosen to develop the assay? Response: Thank you for your careful review and this insightful question. As you say, p54, p72, p22, and other antigens also induce response in pigs. The p30 protein was used as the primary target in our study because it is highly immunogenic and evokes rapid immune response for ASFV early infection in pigs. This is critical for early diagnosis. We mentioned the rationale behind choosing p30 in Lines 316-319. Comment # 7: Line no. 116-117. The reference standard test utilized in this study was ASFV p72 ELISA. However, the assay developed is based on p30 which is not appropriate to make comparison of sensitivity of the assay. Response: Thank you for this question. Based on your comments, we also believe that it inappropriate to use two different methods to compare sensitivity. In order to further verify the sensitivity of our method, we added the gold-standard indirect immunofluorescence assay as the reference method, as detailed in Supplementary Figure S1. Comment # 8: Line no. 141. How the mold and chip has been formed and made into 96 well plate format is not clearly explained. Response: Thank you for this question. Nanoplasmonic sensor chips are manufactured using replication molding technology with molds. The original mold was a tapered nanopillar array (diameter = 180 nm, depth = 450 nm, period = 400 nm) on a silicon wafer made using photolithography and plasma etching. The UV-curable polymer was evenly spread on the mold and placed on a polyethylene terephthalate (PET) sheet to produce the polymeric Nanocup array structure, after which 15 nm of titanium and 60 nm of gold were subsequently deposited on the polymeric Nanocup array in an electron beam evaporator. The sheet was cut to 14 cm × 10 cm and adhered to a 96-well plate with an opening at the bottom or a chip mold produced by a 3D printer (Object 30 prime Stratasys Ltd. USA).
Comment # 9: Line no. 164. What is the detection reagent used for the assay? Response: Thank you for this question. The Nanoplasmonic biosensor platform relies primarily on plasmon resonance sensors with extraordinary light transport (EOT) effects for detection. The principle is based on the effective combination of the ASFV p30 antigen protein labeled with gold nanoparticles and the corresponding antibody in the serum to achieve high-sensitivity detection of ASFV p30 antibodies, which do not require the amplification step of enzyme-linked immunity. Therefore, the detection process only requires PBST buffer containing 10% sucrose and 0.2% Tween 20 (pH = 8.0) as serum diluents.
Comment # 10: Line no.162-175. The principle of binding of the immunological moieties is not clearly explained, even the detection system is not effectively written i.e, chromogenic, luminex. Response: Thank you for this question. The principle of the immunological moieties in the Nanoplasmonic biosensor assay is as follows. The bottom of 3-MPA pre-modified Nanoplasmonic biosensor was coated with goat anti-pig secondary antibody, and after BSA blocking, 50 μL of water was added to read the full spectrum of the chip well. Then, the detected serum and AuNPs-p30 dilution were added. Antigen-antibody complexes were formed among sheep anti-pig secondary antibody, ASFV p30 antibody, and gold particle-labeled p30 protein. The antibody was sandwiched to the surface of the biosensor, resulting in the enhancement of the Surface plasmon resonance (SPR) phenomenon, and the full spectrum OD value of the end point was read at this moment. The displacement difference before and after the detection sample is captured by the Universal microplate reader and read at the special absorbance of 580 nm and 610 nm. Therefore, the system converts the displacement difference generated by antigen antibody complex on the biosensor surface into signal, this detection system is different from chromogenic and luminex. We have added this section to Lines 150-158. Comment # 11: Line 224-255. Authors have not explained the limit of detection of the assay, sensitivity and specificity with reference to gold standard. Response: We are so grateful to you for raising this question. Accordingly, we used gold-labeled indirect immunofluorescence to verify the specificity and sensitivity of our developed method and compared the limit sensitivity of the method. We have added this section to Figure S1 and Figure S2-S12 in the Supplementary Material. Finally, we would like to express our sincere gratitude to the editors and reviewers' for considering our manuscript for publication.
I look forward to hearing from you.